The key metalloprotein of dissimilator nitrite reductase found in the majority of microbial denitrifiers is called cytochrome cd which contains an extractable green heme prosthetic group. This so-called -d-l heme has been assumed to be a chlorin since the late 1950's. Recently a structure determination of isolated heme -d-l by another laboratory also favored a chlorin macrocycle. However, critical analyses of the available spectra data have led us to propose that the -d-1 heme is not a chlorin but a dioxoisobacteriochlorin. Recent experimental studies on demetalated heme -d-1 and model compounds have yielded convincing evidence to support the novel dioxo ring structure. The objectives of the proposed research are (1) to establish unambiguously the structure of the -d-1 heme; (2) to elucidate the intrinsic physicochemical properties of this ring and to identify the attributes which make this heme uniquely suitable for mediating the nitrite reduction; and (3) to understand the reaction pathways and mechanisms. Our experimental approach includes (1) chemical derivatizations of isolated -d-1 for further confirmation of the proposed structure; (2) study of new synthetic methods to prepare dioxoisobacteriochlorins as models and analogues for -d-1 and ultimately, the total synthesis of -d-1; (3) systematic study and documentation of the spectroscopic properties and ligand coordination chemistry of heme -d-1 and model compounds to provide a knowledge base for this class of heme groups; (4) the substitution of synthetic metal dioxo-isobacteriochlorin complexes into the active sites of myoglobin and cytochrome cd to probe the structural basis of prosthetic group/protein interactions; and (5) tests of possible reduction pathways for dioxygen and nitrite reductions using heme models and reconstituted proteins. Denitrification is a basic energy coupled process of metabolism in a large variety of bacteria, including some pathogens. Furthermore, products of denitrification are NO-2-.NO and N20, all of which have direct or indirect toxicity.